Francisella tularensis, the causative organism of tularemia, has long caused illness in animals and, occasionally, in humans. Recently this relatively uncommon human disease has gained new prominence as a potential agent of bioterrorism. Here we propose to study the genetics of F. tularensis to further understand how it grows, causes disease and resists killing by antibiotics. We will utilize methodology we have used to study another intracellular pathogen that causes pneumonia, Mycobacterium tuberculosis. To perform our experiments, we will first develop two new tools. We will construct a DNA microarray representing the predicted genes of F. tularensis and use a transposon to make a highly complex library of F. tularensis mutants. These tools will be combined to compare large pools of mutants simultaneously, allowing us to detect mutants that are unable to survive under specific growth conditions. We will apply this method to three different problems. First, we will search for those F. tularensis genes that are required for in vitro growth. This set of genes contains all of the possible targets for antibiotics. Second, we will identify those genes required for bacterial survival in experimental animals. Knowing these genes should help us understand the molecular mechanisms of F. tularensis pathogenesis. Third, we will define the genes used by F. tularensis to resist many commonly used antibiotics. This could lead to methods to enhance the ability of existing antibiotics to kill the pathogen. Altogether, these studies will help us devise new therapeutic and preventive strategies for tularemia.